Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

16 t h International Congress on Animal Reproduction
Poster Abstracts 199
were used for ES cells establishment (n=18) or differential stained for
cell number counting. All pathenogenetic embryonic stem (pES) cell
lines were characterized by morphology, pluripotency marker of
murine ES cells (AP, Oct-4, SSEA-1, Sox2 and Nanog), chromosome
number as well as in vitro differentiation.
Result The rate of activation and blastocyst formation from MII
oocytes were 90±3.13 and 82.5±2.95%, respectively. The total cell
number, ICM cell number and ICM: trophectoderm ratios of
parthenogenetic blastocysts were 66.8±2.88, 8.1± 0.63 and 1:7.24,
respectively. Four pES cell lines were established and the efficiency
of ES cell lines establishment were 10-37.5%. All pES cell lines
presented a typical morphology of murine ES cell with high nuclear:
cytoplasmic ratio, round shape and clear edge colonies. Three of them
presented normal chromosome number (2n=40). The pES cell lines
possessed high levels of alkaline phosphatase and expressed
pluripotency marker including Oct4, Sox2, SSEA1 and Nanog after
detecting by immunocytochemistry and RT-PCR. During in vitro
differentiation, the pES cell lines formed cystic embryoid bodies in
suspension culture and created spontaneous beating clusters in
gelatine-coated culture dishes.
Conclusion We succeeded to produce ES cell lines from
parthenogenetic blastocysts with presenting pluripotency markers and
normal chromosome numbers in mouse model. This work was
supported by grant from CHE-TRF Senior Scholars, grant No
RTA5080010, The National Research Council of Thailand, The Royal
Thai Government Scholarship (PhD-SW-INV_20060202/49) and EU
FP7 (IAPP 2007 - 218205).
P520
Vitrification versus slow-cooling: higher survival rates for
vitrified two-cell mouse embryos
Temple-Smith, P*; Wang, X; Aguirre Maclennan, I; Momtaz, F; Fage, R;
Patel, D; Philips, S; Pangestu, M; Catt, S
Education Program in Reproduction and Development, Centre for
Reproduction and Development, Monash Institute of Medical Research,
Australia
Introduction Cryopreservation of cleavage stage embryos by slow
cooling is now routine but recently vitrification has gained
prominence. Few studies, however, have compared the two techniques
using the same cohort of embryos.
Here we compare embryo viability in slow-cooled, vitrified or fresh
cultured two-cell mouse embryos (2-CEs).
Methods Oocytes collected from superovulated female mice [F1
(C57Bl6JxCBA)] were fertilised in vitro with epididymal sperm.
Oocytes were washed free of cumulus and sperm and cultured (24h;
modified KSOM, 5% CO2 in air). All 2-CEs (cleavage rate, 84%)
were distributed randomly into 3 groups (slow-cooling, vitrification
and unfrozen controls). For vitrification, 2-CEs were equilibrated (3
min; 37°C) in 10% v/v ethylene glycol, 10% v/v DMSO and placed in
vitrification solution (17% v/v ethylene glycol, 17% v/v DMSO,
0.75M sucrose). Each 2-CE was transferred to a fibreplug (2ul),
touched on a pre-cooled metal block in liquid N 2 and inserted in a precooled
straw (CVM kit, Cryologic). For slow-cooling (SC), up to
seven 2-CEs were put in 11% v/v propanediol in KSOM Hepes (10
mins) followed by 10 min in a final freeze solution [11% v/v
propanediol, 0.5M sucrose in KSOM Hepes medium; room temp
(RT)], and frozen individually in straws using conventional slowcooling
protocols in a programmable freezer (Cryologic CL856).
Vitrified embryos were warmed at 37°C in 3 solutions (0.3M, 0.2M,
0.1M sucrose, 5min in each); SC embryos were thawed at RT in 3
solutions (0.5, 0.25, 0.125M sucrose). Lysis was assessed 2h after
thawing, and all surviving 2-CEs were cultured (72h) to blastocyst
and hatching blastocyst stages assessed. Differences between groups
were examined using a Chi Square test.
Results Lysis rates after thawing were higher for SC embryos (29/113
21.2% cf 4/88 4.5%, P=0.001). Survival and hatching rates after
thawing for vitrification (70/84, 82.6%; 42/84, 50% respectively) and
SC (67/84, 79.7%; 42/84, 50% respectively) groups were not
significantly different from unfrozen controls (73/94, 77.6% and
54/94, 57.7% respectively). However, blastocyst development rate
(67/113, 59.2%) of thawed 2-CE in the SC group was significantly
lower than in the vitrification group (70/88, 79.5%, P=0.002).
Conclusion Comparison of two cryopreservation methods on the
same cohort of cleavage stage embryos revealed lower lysis rates after
vitrification than after slow cooling, but not higher blastocysts rates
from those 2-CE that survived. We suggest that either or both
methods could be used for cryopreserving cleavage stage embryos.
P521
Flat-headed cat cloned embryos and preliminary embryo
transfer
Thongphakdee, A 1 *, Manee-In, S 1 , Rungsiwiwut, R 1 , Numchaisrika, P 1 ,
Siriaroonrat, B 2 , Kamolnorranath, S 2 , Chatdarong, K 1 , Techakumphu, M 1
1Department of Obstetrics Gynaecology and Reproduct, Faculty of Veterinary
Science, Chulalongkorn University, Thailand; 2 Zoological Park Organization
of H.M. the King, Thailand
Introduction Critically endangered flat-headed cat (FC; Prionailurus
planiceps) is one of the small wild cats in Thailand’s captive breeding
program. Inter-generic nuclear transfer (ig-NT) offers the possibility
of FC embryos/offspring production. The purposes of the study were
to evaluate (1) in vitro development and quality of ig-NT FC embryos
(Study 1) and (2) in vivo developmental competence of their transfer
to recipients (Study 2).
Methods In Study 1, 145 ig-NT FC couplets were reconstructed by
fusion of the enucleated in vitro matured (IVM) domestic cat oocyte
together with the starved FC fibroblast cell. The couplets were
activated by inducing electrical pulses, with subsequently incubation
in activation medium, comprised of cycloheximide and cytoclalasin
B, for 4 h. The embryos were cultured in synthetic oviductal fluid
medium supplemented with amino acids and fetal bovine serum, at
38.5°C, 5% CO2 in the humidified atmosphere, and monitored for 7
days. The blastocyst quality was evaluated by cell number count.
Total of 620 IVM cat oocytes were in vitro fertilized (IVF) and served
as control. The cleaved embryos collected at 27 h postinsemination
(pi) were divided for their in vitro development observation in Study 1
(n = 171) and in vivo development after transferred to recipients in
Study 2. The rest of the cleaved embryos were selected for further
study. In Study 2, reconstructed ig-NT FC (n = 135) and IVF cat
embryos (n = 75) were transferred to uterine tubes of gonadotrophintreated
recipients (n = 3 in each group) on Day 2 after hCG-induced
ovulation. Pregnancy was assessed by ultrasonography on Day 30.
Results The reconstructed ig-NT FC couplets were 73.8%
successfully fused. The fused couplets developed to 88.8% cleavage,
44.8% morula and 8.4% blastocyst stages. Oocytes from control IVF
cleaved 54.5% at 27 h pi and those embryos developed to 98% 8-cell,
92% morula and 63% blastocyst stages. The cell number of ig-NT FC
(n = 5) and IVF cat blastocysts (n = 22) was not significantly different
(69±21 vs. 106.4±43). All (3/3) recipients receiving IVF cat embryos
became pregnant and 2 recipients gave to-term kittens, whereas, none
(0/3) receiving ig-NT FC embryos was pregnant.
Conclusions This study establishes the efficiency of ig-NT FC
embryo production. However, developmental ability of ig-NT FC
embryos may be one of the limiting factors of pregnancy
establishment.
P522
Retinol during in vitro fertilization improves development
of mouse embryo
Towhidi, A 1 *, Farshidpour, MR 2 , Chamani, M 3 , Gerami, A 4 , Nouri, M 1
1Animal Science, Islamic Azad University, Shahre Qods Branch, Islamic
Republic of Iran; 2 Animal Science, Islamic Azad University, Varamin Branch,
Islamic Republic of Iran; 3 Animal Science, Islamic Azad University, Science
and Research branch, Islamic Republic of Iran; 4 Statistics, University of
Tehran, Islamic Republic of Iran
Introduction Retinoids are recognized as important regulators of
vertebrate development, cell differentiation, and tissue function.
Pervious studies, were performed both in vivo and in vitro, indicated
the influence of retinoids on several reproductive events, including
follicular development, oocyte maturation and early embryonic